Biochemical and Biophysical Research Communications
Isolated human islets require hyperoxia to maintain islet mass, metabolism, and function
Introduction
Pancreatic islet transplantation is an effective treatment for Type 1 diabetes and allows patients to be free from insulin injections and uncontrollable hypoglycemic episodes [1], [2], [3]. Currently, isolated islets are infused into the liver through the portal vein; however, more than 50% of injected islets are destroyed because of a hostile microenvironment caused by blood-mediated inflammation reactions, low oxygen tension, or high levels of glucose and toxins [3], [4], [5]. Transplantation of isolated islets at extrahepatic sites face similar issues, and these sites often present poor vascularity and hypoxia [6], [7]. Improved oxygenation to the transplantation site might be a promising solution to prevent islet loss; however, the optimal oxygen tension for isolated islets remains unknown.
In the native pancreas, islets are exposed to a high partial oxygen pressure (pO2: approximately 40 mmHg) and receive more blood flow than the rest of the pancreatic tissue [3], [7], indicating that islets consume more oxygen because of their high metabolic activity [6]. Unlike whole organ transplants which largely preserve capillaries, islets used for transplantations are isolated by enzymatic and mechanical digestion which destroys the islet capillary network [8]. According to current practices, after isolation islets are cultured for a short period under 21% oxygen and 5% CO2 at one atmospheric pressure, and the pO2 of the media is equilibrated to a predicted 160 mmHg. Equilibration and the predicted pO2 value are calculated mathematically without considering the oxygen consumed by active islets. Although the predicted pO2 is four times higher than that reported in the native pancreas, islet central necrosis often develops. This suggests that the oxygen requirement for isolated islets differs from that in the native pancreas possibly due to environmental or structural changes. In this regard, it remains critically important to determine the optimal pO2 that maintains the health of isolated islets pre-transplantation.
Providing the optimal pO2 is also crucial post-transplantation. Regardless of the transplant sites, islets have to rely on the oxygen within surrounding tissue until newly formed microcapillaries can begin to deliver oxygen [3], [7], [9]. However, similar to cultured islets, there is no available information that quantifies the pO2 required for islets to survive post-transplantation. Keeping in mind the need to establish evidence based guidelines, the following in vitro studies were conducted to determine the optimal oxygen concentration for isolated human islets and to identify pO2 levels that promote improved transplantation efficiencies and better patient outcomes.
Section snippets
Isolation of human islets
Human islets were isolated by the Islet Manufacturing Team of the Southern California Islet Cell Resources Center (SC-ICRC) [8], [10]. The use of human tissues in this study was approved by the Institutional Review Board of the Beckman Research Institute.
Human islet culture under different oxygen concentrations
Islets were cultured in a 24-well plate at 250 IEQ/well. Cells were maintained in 1000 μL of RPMI1640 medium containing 5 mmol/L glucose and 10% FBS at 37 °C for 7 days under various oxygen concentrations (10, 21, 35, and 50% oxygen) plus 5% CO2
Simulated and measured values of pO2 in culture medium under various oxygen settings
Simulation results predicted a pO2 gradient within the media as a result of islet oxygen consumption. The simulated pO2 around islets under air containing 21% oxygen were calculated as 152.6, 147.4 and 134.3 mmHg in 300, 500 and 1000 μL of media, respectively (Fig. 1A). In the simulation, OCR also acted to decrease the pO2 (Fig. 1B). The measured average pO2 in 1000 μL of culture media on day 1 under 10, 21, 35 and 50% oxygen was 90, 160, 270 and 350 mmHg, respectively (Fig. 1C).
Cultured islets gradually degrade under 21% oxygen
The physical
Discussion
To date, we have had little direct knowledge of how pO2 influences islet physiology and survival and how these changes affect islet transplantation efficiency. Current practices culture isolated human islets under normoxic conditions (21% oxygen) before transplantation. In contrast, we find that islets cultured under hyperoxic conditions better maintain islet volume, viability, metabolism, and function.
Our simulations imply that culture methods considerably affect pO2 around the islets, which
Acknowledgments
H.K. designed the study, collected data, and wrote the manuscript. D.K., L.M., A.B. and D.M. collected data. J.R., K.O, K.F., Y.T., F.K. and Y.M. reviewed and edited the manuscript.
This work was supported by a grant from the Nora Eccles Treadwell Foundation (30.6990.973667). Human islets were provided by the Integrated Islet Distribution Program (IIDP). We acknowledge the Manufacturing Team led by Dr. Ismail Al-Abdullah at the Southern California Islet Cell Resources Center, Beckman Research
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